1. Field of the Invention
The present invention is in the field of compositions comprising vesicles, such as liposomes and outer membrane vesicles (OMVs) obtainable from Gram negative bacteria, methods of making such compositions and vaccines based thereon. In particular, the invention relates to vaccines and pharmaceutical compositions comprising OMVs obtained from Neisseria species.
2. Description of the Related Art
A significant number of human and animal pathogens fall within the Gram negative classification of bacteria, including members of the genus Neisseia, Moraxella, Kingella, Acinetobacter, Brucella, Bordetella, Haemophilus, Escherichia, Chlamydia, Legionella, Pseudomonas, Proteus and Yersinia. Neisseria meningitidis (the meningococcus) is the organism that causes meningococcal meningitis and is of particular importance as a worldwide health problem. In many countries the incidence of this disease is increasing. N. meningitidis is also responsible for meningococcal septicaemia, which is associated with rapid onset and high mortality, with around 22% of cases proving fatal. Other Gram negative bacteria are responsible for a range of human infections including meningitis (H. influenzae), plague (Y. pestis), gastroenteritis (E. coli), venereal disease (N. gonorrhoeae) and nosocomial infection (P. aeruginosa).
It would be desirable to provide alternative broad spectrum vaccines that provide protective immunity in animals, particularly humans, against Gram negative bacterial infection, and especially infection by Gram negative pathogens.
Many known vaccines are based upon preparations of capsular polysaccharide, however, these vaccines are often limited in their protective value. For example, vaccines directed at providing protective immunity against meningococcal disease provide only limited protection because the protection tends to be strain specific whereas there are many different strains of N. meningitidis. Vaccines based upon the serogroup antigens, the capsular polysaccharides, offer only short lived protection against infection and do not protect against many strains commonly found in North America and Europe. In fact, certain capsular polysaccharides, such as that from the group B meningococcal capsule, are essentially non-immunogenic in humans.
The outer membrane of many Gram negative bacteria is highly dynamic and can produce vesicles that bud off and are released into the surrounding environment. These outer membrane vesicles (OMVs), also referred to as blebs, comprise many of the outer membrane proteins (OMPs) and lipopolysaccharide (LPS) that contribute to the antigenic profile of the organism.
There have been a number of attempts to generate an OMV based vaccine in the hope that it could overcome the disadvantages seen in previous capsular polysaccharide based vaccines. In Bjune et al. (Lancet (1991) 338: pp 1093-1096) a vaccine consisting of OMVs from group B N. meningitidis is described (Norwegian vaccine). Bjune et al. show that the vaccine was able to induce a protective efficacy against meningococcal disease of 57.2% in a clinical trial in Norway. A similar vaccine has been produced in Cuba (Sierra et al., NIPH Ann (1991) December;14(2): pp 195-207) and high levels of efficacy were observed in that country. However, a large study in Brazil showed poor efficacy of the Cuban vaccine, especially in young children (de Moraes et al., Lancet (1992) October 31;340(8827): pp 1074-1078).
To address the difficulties associated with achieving broad spectrum protection researchers have attempted to “enrich” OMVs with particular antigens that might enhance the immunogenic potential of the OMV. In WO-A-00/25811 OMVs isolated from N. meningitidis are combined with heterologous antigens, e.g. Tbp, or a genetically modified N. meningitidis expresses such antigens recombinantly and antigen enriched OMVs are derived therefrom. A similar approach was adopted by researchers in WO-A-01/09350 which describes vaccine compositions comprising OMVs from N. meningitidis, M. catarrhalis and H. influenzae, where in certain embodiments these organisms have been genetically modified to overexpress particular immunogenic moieties.
A further OMV based vaccine composition is known as the Hexamen™ or Dutch vaccine (Cartwright et al., Vaccine 17 (1999), pp 2612-2619). The Hexamen™ vaccine composition comprises N. meningitidis OMVs that include six different PorA proteins that are recombinantly produced using two vaccine strains of N. meningitidis, PL16215 and PL10124. Each strain is capsule negative and produces three different PorA proteins, CPS−P1.7,16;P1.5,2; P1.19,15 and CPS−P1.5c,10; P1.12,13; P1.7h,4 respectively.
Ruppe Van der Voort et al (Vaccine (2000) 18(14); pp 1334-1343) show that the hexamen vaccine induces specific serum bactericidal antibodies against all six PorA sero-subtypes included in the vaccine. However, the Hexamen™ vaccine suffers from certain drawbacks. It is not currently possible to express all six PorA proteins in a single N. meningitidis cell due to host toxicity problems. Hence, three PorAs are expressed in one cell and three in another. OMVs from cell cultures each expressing three PorAs are obtained and then admixed to give the hexavalent vaccine.
PorA is an immunodominant antigen, meaning that it masks most other antigens presented on the OMV surface—i.e. Hexamen™ vaccine is heavily biased towards PorA as the protective antigen. This, in turn, leads to selection pressure in the population towards strains of N. meningitidis that express PorAs that are antigenically different to those in the Hexamen™ vaccine. There is, therefore, a risk that strains of N. meningitidis that are not protected against by the Hexamen™ vaccine will predominate in time, resulting in ongoing efforts to continually modify the vaccine to protect against strains currently infecting and causing disease in the human population.
It would be desirable to provide an OMV vaccine composition that provides broad spectrum protection to infection from a number of bacterial species and at least a wide range of strains within a single bacterial genus. It would be particularly desirable to produce an OMV-based vaccine that provides broad spectrum, long term protection against disease caused by a range of strains of Gram negative bacteria, and especially N. meningitidis. 
It would further be desirable to provide further and/or improved methods of preparing vesicle-containing compositions.